The cannabinoid receptor 1 (CB1) is a G-protein coupled receptor (GPCR) that regulates neural transmission and other physiological processes. Considerable excitement surrounds the therapeutic potential of CB1 including to regulate appetite, for the relief of neuropathic pain, and to prevent relapse to the use of drugs of abuse. CB1 is activated by the endogenous ligands arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) and various synthetic and plant-derived ligands (e.g. 9 tetrahydrocannabinol; THC). These CB1 agonists transduce signals to downstream effectors primarily via Gi/o coupling. Moreover, since CB1 also exhibits ligand-independent, constitutive activity, compounds able to block this activity provide vital tools to probe the profile of constitutive levels of signal transduction and the consequence of their inhibition. Furthermore, recent evidence suggests that pregnenolone, a CB1 ligand with inverse agonist activity, is elevated following prolonged THC use illustrating an endogenous mechanism to counterbalance CB1 over- activity. The goal of this project is to profile the molecular-level signal transduction patterns of the basally active and inactive forms of CB1 induced by inverse agonists including first generation, endogenous, and novel compounds, to establish biased-inverse agonism for the first time. While biased agonism is now well recognized and changing the GPCR drug discovery landscape, biased inverse agonism has not been examined. CB1 is an ideal candidate to address this issue and this is critical knowledge for the longer-term goal of developing these compounds for the treatment of overeating behaviors and addiction. In this project, we will (1) contrast CB1 constitutive activity and CB1 inactive forms using inverse agonists of diverse structure. We will identify the downstream CB1 signaling patterns in the presence and absence of robust inverse agonists using global arrays to identify alterations in kinase phosphorylation, mRNA, and miRNA expression (critical for mRNA regulation) in basally active and inverse agonist treated cells. CB1 activity will be correlated with subcellular localization of the receptor in its basal and differentially inactive forms. We will (2) exploit ne benzhydrylpiperazine and coumarin scaffolds to yield novel potent and chemically diverse CB1 inverse agonists. We will synthesize these derivatives to increase potency and characterize their signal transduction patterns. Recently, we identified an inverse agonist of CB1 with a Ki = 220 nM that lacks the heteroaromatic central core common to CB1 inverse agonists. This new scaffold presents the opportunity to develop peripherally active CB1 inverse agonists for treating obesity without the unwanted anxiogenic and depressogenic side effects associated with centrally active CB1 inverse agonists. By characterizing a structurally diverse set of inverse agonists, differential effects on cell responses will be observed to guide the development of future therapeutics.